The goals of this research project are to elucidate some of the biochemical mechanisms by which an RNA-enveloped virus is replicated and released from its animal cell host. Tissue culture cells infected with Sindbis virus and cell-free protein synthesizing systems provided with two Sindbis viral-specific messenger RNAs will provide the experimental systems for identifying viral-specific proteins and for isolating and studying the proteolytic enzymes required for processing these proteins from large molecular weight precursor polypeptides that appear as the initial viral gene products. Mutationally-altered forms of Sindbis virus, metabolic and protease inhibitors, different host cells and different growth conditions are to be used (1) for accumulating, isolating and identifying precursors and products that form the viral-specific replicase and the virion structural proteins, and (2) for studying the regulation of viral gene expression and viral RNA formation during replication. Results of these studies should provide information on how viral gene products may control viral replication, the extent to which host cell enzymes may control viral growth, and the role that virus may play in modifying host cell enzymes. One of the essential steps in the maturation of Sindbis virus consists of a proteolytic cleavage of a viral glycoprotein that forms part of the host cell plasma membrane. There is increasing evidence that alteration of glycoproteins on cell surfaces may be an important event in the regulation of animal cell growth and metabolism. Thus, the research proposed here on modifications of viral membrane proteins should not only reveal viral-cell relationships but may also provide a valuable model system for probing regulation of normal cell growth.